Reconstitution of water channel function of aquaporins 1 and 2 by expression in yeast secretory vesicles.
Aquaporins 1 (AQP1) and 2 (AQP2) were expressed in the yeast secretory mutant sec6-4. The mutant accumulates post-Golgi, plasma membrane-targeted vesicles and may be used to produce large quantities of membrane proteins. AQP1 or AQP2 were inducibly expressed in yeast and were localized within isolated sec6-4 vesicles by immunoblot analysis. Secretory vesicles containing AQP1 and AQP2 exhibited high water permeabilities and low activation energies for water flow, indicating expression of functional AQP1 and AQP2. AQP1 solubilized from secretory vesicles was successfully reconstituted into proteoliposomes, demonstrating the ability to use the yeast system to express aquaporins for reconstitution studies. The AQP2-containing secretory vesicles showed no increased permeability toward formamide, urea, glycerol, or protons compared with control vesicles, demonstrating that AQP2 is highly selective for water over these other substances. We conclude that the expression of aquaporins in yeast sec6 vesicles is a valid system to further study mammalian water channel function.
Coury
L A
LA
Department of Medicine, University of Pittsburgh Medical Center, Pennsylvania 15213-2500, USA.
Mathai
J C
JC
Prasad
G V
GV
Brodsky
J L
JL
Agre
P
P
Zeidel
M L
ML
D051398:Aquaporin 1; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins; D001789:Blood Group Antigens; D003001:Cloning, Molecular; D003594:Cytoplasmic Granules / Q000502:physiology*; D005559:Formamides / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D006801:Humans; D007473:Ion Channels / Q000096:biosynthesis / Q000302:isolation & purification / Q000502:physiology*; D007700:Kinetics; D008970:Molecular Weight; D010539:Permeability; D011522:Protons; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology*; D013816:Thermodynamics; D014508:Urea / Q000378:metabolism
C496020:AQP1 protein, human; C496023:AQP2 protein, human; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins; D001789:Blood Group Antigens; D005559:Formamides; D007473:Ion Channels; D011522:Protons; D011994:Recombinant Proteins; D051398:Aquaporin 1; C031066:formamide; D014508:Urea; D005990:Glycerol